Student University of Texas at Arlington Arlington, Texas, United States
Introduction:: Divergence in the metabolic pathways provided by bacteria in the gut can lead to differences in the structure of dietary compounds that are ultimately absorbed by the host and in turn influence health. One example is the microbially synthesized metabolite equol formed from the dietary isoflavone daidzein found in soy. Equol is known as a selective agonist for estrogen receptor beta signalling and has been indicated in reducing cardiovascular disease risk. A fraction of the population have the intestinal bacteria capable of converting daidzein from dietary soy into equol. Individuals with an equol producing phenotype are reported to have a range of health benefits with daidzein intake, including reduced larger artery stiffness. This isoflavone conversion pathway at its impact on human health prompted us to examine a means to engineer the intestinal microbiota toward an equol producing phenotype by introducing genes to the gut bacteria for expression of the enzymes daidzein reductase (DR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR). Here we provide evidence of an engineered bacterial metabolic pathway for enzymatic conversion of the isoflavone daidzein to equol, and moreover demonstrate its ability to increase serum equol levels in an in vivo model provided daidzein.
Materials and Methods:: A set of plasmids were first constructed to facilitate expression of the enzymes DR, DHDR, and THDR derived from a healthy human gut described in literature to have high activity for daidzein conversion to equol. The gene products DR, DHDR, and THDR were expressed in E. coli and the cultures were provided varying concentrations of daidzein in order monitor its conversion. Production of dihydrodaidzein, tetrahydrodaidzein, and equol (the desired end product) were assessed qualitatively by thin layer chromatography of the extracted isoflavones and monitored quantitatively by liquid chromatography – mass spectrometry as a function of time. The engineered E. coli and controls were provided to mice undergoing different diets with and without daidzein to assess the impact on serum equol levels. Fecal pellets were also assessed to examine the impact of the diet and probiotic on the bacterial diversity of the gut and were assessed by 16s metagenomics analysis.
Results, Conclusions, and Discussions:: We found that the enzyme gene products of DR, DHDR, and THDR to be necessary and sufficient for conversion of daidzein to equol. In assessing plasmid persistence in the murine gut through the use of probiotic bacteria as carriers of genetic constructs and their ability to persist over a controlled timeframe in the intestine, we found the residence time is impacted by dosage factors as well as selection pressure for retention of the gene constructs. The probiotic bacteria carriers of the gene constructs for DR, DHDR, and THDR were found to be effect in increasing the serum levels of equol thus confirming our ability to change the host gut activity to that of an equol producing phenotype. The impact of this work thus shows this to be an effective means for modification or introduction of metabolic pathways in the gut, wherein we can use such a probiotic approach to modify the biochemical profile from consumed foods and in doing so lead to improved health. We are hopeful that this approach may provide an acceptable alternative to fecal transplant therapy for future needs in modifying metabolic pathways of the gut, as we show here this to be a promising approach to engineering the intestinal microbiota toward an equol producing phenotype.
